Homonuclear Scalar Couplings Research Articles

Apparent Transverse Relaxation Rates in Systems with Scalar-Coupled Protons

The modulation of spin echoes by homonuclear scalar couplings render the determination of transverse relaxation rates of individual spins difficult, in particular for molecules that are isotopically enriched in (13)C or (15)N, and for all molecules with scalar-coupled protons. To avoid echo modulations, most studies using refocusing pulses have so far been restricted to isolated (1)H, (13)C, or (15)N spins. We report measurements of apparent (1)H transverse relaxation rates of backbone and side-chain protons in Cyclosporin A (CsA) determined by quenching the echo modulations that arise from homonuclear scalar couplings between protons.

J-Based 3D sidechain correlation in solid-state proteins.

Scalar-based three-dimensional homonuclear correlation experiments are reported for (13)C sidechain correlation in solid-state proteins. These experiments are based on a sensitive constant-time format, in which homonuclear scalar couplings are utilized for polarization transfer, but decoupled during chemical shift evolution, to yield highly resolved indirect dimensions and band selectivity as desired. The methods therefore yield spectra of high quality that give unique sets of sidechain correlations for small proteins even at 9.4 Tesla (400 MHz (1)H frequency). We demonstrate versions of the pulse sequence that enable correlation from the sidechain to the backbone carbonyl as well as purely sidechain correlation sets; together these two data sets provide the majority of (13)C-(13)C correlations for assignment. The polarization transfer efficiency is approximately 30% over two bonds. In the protein GB1 (56 residues), we find essentially all cross peaks uniquely resolved. We find similar efficiency of transfer (approximately 30%) in the 140 kDa tryptophan synthase (TS), since the relaxation rates of immobilized solid proteins are not sensitive to global molecular tumbling, as long as the correlation time is much longer than the magic-angle spinning rotor period. In 3D data sets of TS at 400 MHz, some peaks are resolved and, in combination with higher field data sets, we anticipate that assignments will be possible; in this vein, we demonstrate 2D (13)C-(13)C spectra of TS at 900 MHz that are well resolved. These results together provide optimism about the prospects for assigning the spectra of such large enzymes in the solid state.

Determination of transverse relaxation rates of individual spins while quenching echo modulations due to homonuclear scalar couplings

The envelopes of spin-echo amplitudes obtained with trains of multiple refocusing pulses are normally modulated by homonuclear scalar couplings. Echo modulations make it difficult to determine transverse relaxation rate constants R 2. Provided that the pulses are of moderate amplitude so that spins at different offsets experience different tilted effective fields, and provided one avoids recoupling conditions, it is possible to quench the modulations of spin echoes. When the modulations are quenched, apparent transverse relaxation rate constants R 2( I) can be determined for individual spins I. The principle is demonstrated for solutions of glycine with single or double carbon-13 enrichment.

Quenching and Recoupling of Echo Modulations in NMR Spectroscopy

Trains of spin echoes are normally modulated by homonuclear scalar couplings. It has long been known that echo modulations are quenched when the pulse-repetition rates are much larger than the offsets of the coupling partners, because the spin systems behave as if they consisted of magnetically equivalent spins when the offsets are suppressed. This type of quenching of the echo modulations can occur when the radio-frequency (RF) pulses are ideal, that is, when they are perfectly homogeneous, properly calibrated to induce rotations through an angle, pi, and have an RF amplitude, omega(1)=-gammaB(1), that is strong compared to the largest offset, Omega(S)=omega(0S)-omega(RF), with respect to the carrier frequency. Recently, it was discovered that echo modulations can also be quenched when the RF pulses are nonideal, that is, when they are too weak to bring about an ideal rotation of the magnetization of the coupling partners, so that the effective fields associated with the RF pulses are tilted in the rotating frame. This phenomenon typically occurs when the pulse-repetition rates are much slower than the offset of the coupling partner. Under such conditions, it turns out, however, that for certain offsets, when the phase, Phi(S) (which arises from a free precession of the magnetization of the coupling partner, S, in the pulse interval, 2tau, and the pulse length, tau(pi)), approaches a multiple of 2pi, the echo modulations are restored. However, the frequencies of these echo modulations are not simply determined by the homonuclear scalar coupling, J(IS). The Fourier transforms of the echo trains (the so-called "J spectra") reveal surprising multiplet patterns, and the amplitudes of the echo modulations depend on the offsets of the coupling partners. Herein, we present a unified theory, based on an average-Hamiltonian approach, to describe these effects for two-spin systems. Experimental evidence of echo modulations in a system of two spins is presented. Experiments with three and more spins, backed up by extensive numerical simulations, will be presented elsewhere.

Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Cα and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-(13)C]- or [2-(13)C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C(alpha) sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta1) are produced with isolated (13)C spins (i.e., no (13)C-(13)C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C(beta) positions for 17 of the common 20 amino acids and there are no cases for which (13)C(alpha)-(13)CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone (15)N studies. The utility of the labeling is established by recording (13)C R (1rho) and CPMG-based experiments on a number of different protein systems.

Double-quantum filtered rotational-echo double resonance

The homonuclear scalar coupling of a directly bonded 13C– 13C pair has been used to create a double-quantum filter (DQF) to remove the natural-abundance 13C background in 13C{ 15N} rotational-echo double-resonance (REDOR) experiments. The DQF scalar and REDOR dipolar evolution periods are coincident which is important for sensitivity in the event of weak 13C– 15N dipolar coupling. Calculated and observed 13C{ 15N} DQF–REDOR dephasings were in agreement for a test sample of mixed recrystallized labeled alanines. Glycine metabolism in a single uniform- 15N soybean leaf labeled for 6 min by 13CO 2 was measured quantitatively by 13C{ 15N} DQF–REDOR with no background interferences.

Optimized detection of lactate at high fields using inner volume saturation

In localized proton MR spectroscopy ((1)H-MRS) in vivo, the detection of lactate (Lac) is affected by modulation of its resonances due to homonuclear scalar couplings (J). A simple and convenient way to distinguish Lac from lipids is to set the TE to 1/J so that the Lac signal is inverted while other resonances (such as lipid) remain in-phase. However, at high field strengths, such as 3 Tesla or above, the modulation of the Lac signal is complicated by chemical shift effects that cause modulation patterns to vary within different subregions of the localized volume. Under some conditions the Lac signal may even disappear completely. In this note we introduce the concept of inner volume saturation (IVS), which makes use of high bandwidth spatial pulses to remove the signal corresponding to the regions of the localized volume that contribute unwanted modulation patterns. The method is described theoretically and demonstrated experimentally at 3 Tesla in a phantom and a patient with acute stroke. The phantom measurements indicate that virtually 100% of the Lac signal can be recovered using this method. The method should be feasible at magnetic fields above 3 Tesla, and may also be applied to other coupled spin systems in which modulation effects are important.

Selective NMR Measurements of Homonuclear Scalar Couplings in Isotopically Enriched Solids

Scalar (J) couplings in solid-state NMR spectroscopy are sensitive to covalent through-bond interactions that make them informative structural probes for a wide range of complex materials. Until now, however, they have been generally unsuitable for use in isotopically enriched solids, such as proteins or many inorganic solids, because of the complications presented by multiple coupled but nonisolated spins. Such difficulties are overcome by incorporating a z-filter that results in a robust method for measuring pure J-coupling modulations between selected pairs of nuclei in an isotopically enriched spin system. The reliability of the new experimental approach is established by using numerical simulations and tested on fully (13)C-labeled polycrystalline L-alanine. It is furthermore shown to be applicable to partially enriched systems, when used in combination with a selective double-quantum (DQ) filter, as demonstrated for the measurement of (2)J((29)Si-O-(29)Si) couplings in a 50% (29)Si-enriched surfactant-templated layered silicate lacking long-range 3D crystallinity. J-coupling constants are obtained with sufficient accuracy to distinguish between different (29)Si-O-(29)Si pairs, shedding insight on the local structure of the silicate framework. The new experiment is appropriate for fully or partially enriched liquid or solid samples.

Quenching Echo Modulations in NMR Spectroscopy

In NMR spectroscopy, homonuclear scalar couplings normally lead to modulations of spin echoes that tend to interfere with the accurate determination of transverse relaxation rates by Carr-Purcell-Meiboom-Gill (CPMG) multiple refocusing experiments. Surprisingly, the echo modulations are largely cancelled when the refocusing pulses applied to the coupling partner deviate slightly from ideal pi rotations due to tilted effective radio-frequency (RF) fields, even at offsets that are much smaller than the radio-frequency amplitude. Experiments and simulations illustrate these effects for two-spin IS systems containing donor and acceptor (15)N nuclei I=N (D) and S=N(A) in RNA Watson-Crick base pairs with homonuclear scalar couplings J(IS)=(2h)J(N(D), N(A)) across the hydrogen bonds.

PFG NMR diffusion experiments for complex systems

Many practical applications of diffusion NMR, ranging from biomedical to industrial, entail the measurement of low-concentration solutes in non-deuterated, compositionally complex systems. The aim of this article is to present examples of robust, versatile diffusion experiments that can be used with non-deuterated solvents and at non-ambient temperatures. Specifically, three experiments are presented in detail: CONVEX, which combines excitation-sculpting solvent suppression with double-echo convection-compensating PGSE; DQDiff, which implements double-quantum filtered diffusion measurements in a convection-compensating mode; and applications of Oscillating-Gradient Spin-Echo (OGSE) to systems with homonuclear scalar couplings. These examples are based on the recent work by the authors and relate to a variety of systems, ranging from simple solutions to colloidal and polymeric systems. Besides the applied aspects, we review the general methodology used to treat the effects of diffusion and flow in NMR experiments, and apply this theory to derive the diffusion attenuation expression for each of the experiments presented. The paper should be useful to beginners as well as advanced users of general NMR wishing to learn about diffusion measurements.

Solid‐State Magic‐Angle Spinning NMR of Outer‐Membrane Protein G from Escherichia coli

Uniformly 13C-,15N-labelled outer-membrane protein G (OmpG) from Escherichia coli was expressed for structural studies by solid-state magic-angle spinning (MAS) NMR. Inclusion bodies of the recombinant, labelled protein were purified under denaturing conditions and refolded in detergent. OmpG was reconstituted into lipid bilayers and several milligrams of two-dimensional crystals were obtained. Solid-state MAS NMR spectra showed signals with an apparent line width of 80-120 Hz (including homonuclear scalar couplings). Signal patterns for several amino acids, including threonines, prolines and serines were resolved and identified in 2D proton-driven spin-diffusion (PDSD) spectra.

Acquisition of pure-phase diffusion spectra using oscillating-gradient spin echo

Oscillating-gradient spin echo (OGSE) diffusion experiments have long been used to measure the short-time apparent diffusion coefficient, D app ( t), in the presence of restricted diffusion, as well as the spectrum of the slow-motion velocity autocorrelation function. In this work, we focus on two previously unexplored aspects of OGSE experiments: convection compensation and acquisition of pure-phase diffusion spectra in the presence of homonuclear scalar couplings. We demonstrate that convection compensation afforded by single-echo OGSE compares well with that in double-echo convection-compensated PGSE experiments. We also show that, in the presence of homonuclear scalar couplings, setting the OGSE echo time to 1/2 J enables acquisition of pure-phase diffusion spectra and yields more reliable D estimates than mixed-phase PGSE or OGSE spectra. Pure-phase OGSE acquisition is also compatible with measurements of the apparent diffusion coefficient at an arbitrary diffusion time. These features of OGSE can be valuable in diffusion measurements of scalar-coupled small-molecule probes in cellular and other heterogeneous systems.

Resolution enhancement in MAS solid-state NMR by application of 13C homonuclear scalar decoupling during acquisition

Spectral resolution imposes a major problem on the evaluation of MAS solid-state NMR experiments as larger biomolecular systems are concerned. We show in this communication that decoupling of the 13C–13C homonuclear scalar couplings during stroboscopic detection can be successfully applied to increase the spectral resolution up to a factor of 2–2.5 and sensitivity up to a factor of 1.2. We expect that this approach will be useful for the study of large biomolecular systems like membrane proteins and amyloidogenic peptides and proteins where spectral overlap is critical. The experiments are demonstrated on a uniformly 13C,15N-labelled sample of Nac-Val-Leu-OH and applied to a uniformly 13C,15N-enriched sample of a hexameric amyloidogenic peptide.

Determination of (1)H homonuclear scalar couplings in unlabeled carbohydrates.

The scarcity of structural information on carbohydrates results from combined difficulties to crystallize and the limitations in NMR analysis. Current methods for determining basic NMR parameters such as (1)H homonuclear scalar couplings are very limited, especially for large molecules such as polysaccharides, oligonucleotides, and the carbohydrate part of glycoproteins. In this paper, a NMR experiment for the determination of endocyclic (1)H homonuclear scalar couplings ((3)J(HH)) in unlabeled carbohydrates is presented. In addition to scalar couplings, cross-correlated dipole-dipole relaxation rates were measured for large polysaccharides. The measurement of all endocyclic homonuclear coupling constants within monosaccharide units is presented for lactose, a model disaccharide, and for a natural-abundance 2 MDa bacterial polysaccharide excreted by Streptococcus thermophilus Sfi39.

Refined structure of a flexible heptasaccharide using 1H-13C and 1H-1H NMR residual dipolar couplings in concert with NOE and long range scalar coupling constants.

The heptasaccharide isolated from the cell wall polysaccharide of Streptococcus mitis J22 serves as an important model for the dynamics and conformation of complex polysaccharides, illustrating the nature of flexibility with rigid epitopes joined by flexible hinges. One-bond C-H residual dipolar couplings (1D(CH)) and long-range H-H residual dipolar couplings (nD(HH)) were measured for the heptasaccharide in a cetylpyridinium chloride/hexanol/brine lamellar liquid crystal medium. A method is proposed to determine the nD(HH) in natural abundance based on a 13C resolved 1H TOCSY pulse sequence previously published to determine the homonuclear scalar couplings. Different methods for interpretation of the 1D(CH) and the nD(HH) residual dipolar coupling data obtained were compared and combined with the NOE and long-range H,C and C,C scalar couplings available for this heptasaccharide. A flexible model of the heptasaccharide was determined in which two structurally well-defined regions involving four and two sugar residues, respectively are joined by a flexible hinge which involves two 1-->6 glycosidic linkages.

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings.

A modified version of the JHH-TOCSY experiment, 'signed COSY', is presented that allows the determination of the sign of residual dipolar 1H-1H coupling constants with respect to the sign of one-bond 1H-X coupling constants in linear three-spin systems X-1H-1H, where X = 13C or 15N. In contrast to the original JHH-TOCSY experiments, the signs of J(HH) couplings may be determined for CH2-CH2 moieties and for uniformly 13C/15N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.

Determination of Homonuclear Scalar Coupling Constants by Combining Selective Two-DimensionalNMR Spectroscopy with Convolution and Deconvolution: Applications to Paclitaxel (Taxol)

Homonuclear proton-proton coupling consts. can be detd. accurately by recording selective two-dimensional correlation spectra and by analyzing the resulting multiplets by two-dimensional convolution and deconvolution methods. These methods were evaluated by attempting to det. the complete set of homonuclear couplings in paclitaxel (taxol). Convolution methods have been applied successfully to ten pairs of complementary multiplets recorded by selective correlation spectroscopy (soft-COSY) and pure in-phase correlation spectroscopy (PICSY). These pairs of multiplets gave reliable measurements of ten active couplings. In some cases, satisfactory PICSY spectra could not be obtained, so that the complementary information of soft-COSY and PICSY could not be exploited. Furthermore, couplings involving two strongly-coupled diastereotopic protons (14Ha and 14Hb) could not be detd. in satisfactory manner. Further problems arose due to partial overlap of the chem. shifts of two other protons (10H and 13H). Deconvolution has been applied successfully to all 14 soft-COSY multiplets, yielding 14 ests. of active couplings and 38 ests. of passive splittings. This work provides between one and seven ests. for 21 coupling consts. The accuracy of these different ests. must be weighed with the complexity of the methods used. This work may help to assess the perspectives of completely automated computer-supported anal. of two-dimensional spectra. [on SciFinder (R)]

Theoretical treatment of volume localized NMR spectroscopy in weakly coupled spin systems

A pulse sequence is proposed for obtaining volume selective echoes with both chemical shift and homonuclear scalar coupling refocused. Theory, in terms of product operator formalism, is presented and the full response of the spin system is computed. The theoretical spatial dependence of the power signal given by spins localized in different planes (x=0,y=0, respectivez=0) is presented.

A pulse sequence for averaging the strong homonuclear scalar coupling in AB spin systems

A pulse sequence is proposed for the suppression of the strong homonuclear scalar coupling in the case of AB spin systems. The theoretical treatment is presented in terms of the average Hamiltonian theory in the case of aperiodic perturbations. The zero-order and the first-order correction terms in the full average Hamiltonian are calculated. It is shown that the chemical shift interaction for one of the spins is completely refocused and the conditions in which the interactions bilinear in spin operators are efficiently suppressed are analyzed.

Detection of topologies in homonuclear pseudo-3D NMR

A general three-period homonuclear NMR experiment is analyzed in terms of the minimal scalar coupling topologies which can be detected. It is shown that the number of such minimal topologies is small, that specific topologies can be selected by appropriate choice of pathway types and quantum orders in the experiment, and that in experimental spectra each resonance can be ascribed to a particular minimal topology. The information content of 3D multiple-quantum spectroscopy and of many common homonuclear 2D NMR experiments is analyzed in terms of whether a given minimal topology is observable and can be interpreted. The use of pseudo-3D multiple-quantum spectroscopy as a method for automated identification of homonuclear scalar coupling topologies is explored using the computer programs Predictor, Equivalent, and Comparator.